Amifostine induces antioxidant enzymatic activities in normal tissues and a transplantable tumor that can affect radiation response

Abstract

Purpose: To determine whether amifostine can induce elevated manganese superoxide dismutase (SOD2) in murine tissues and a transplantable SA-NH tumor, resulting in a delayed tumor cell radioprotective effect.

Methods and materials: SA-NH tumor-bearing C3H mice were treated with a single 400 mg/kg or three daily 50 mg/kg doses of amifostine administered intraperitoneally. At selected time intervals after the last injection, the heart, liver, lung, pancreas, small intestine, spleen, and SA-NH tumor were removed and analyzed for SOD2, catalase, and glutathione peroxidase (GPx) enzymatic activity. The effect of elevated SOD2 enzymatic activity on the radiation response of SA-NH cells was determined.

Results: SOD2 activity was significantly elevated in selected tissues and a tumor 24 h after amifostine treatment. Catalase and GPx activities remained unchanged except for significant elevations in the spleen. GPx was also elevated in the pancreas. SA-NH tumor cells exhibited a twofold elevation in SOD2 activity and a 27% elevation in radiation resistance. Amifostine administered in three daily fractions of 50 mg/kg each also resulted in significant elevations of these antioxidant enzymes.

Conclusions: Amifostine can induce a delayed radioprotective effect that correlates with elevated levels of SOD2 activity in SA-NH tumor. If limited to normal tissues, this delayed radioprotective effect offers an additional potential for overall radiation protection. However, amifostine-induced elevation of SOD2 activity in tumors could have an unanticipated deleterious effect on tumor responses to fractionated radiation therapy, given that the radioprotector is administered daily just before each 2-Gy fractionated dose.

Fig. 1

Comparative DNA histograms of cells isolated from spleens and SA-NH tumors in C3H mice as determined by flow cytometry.

Fig. 2

The effects of a single 400 mg/kg dose of amifostine on manganese superoxide dismutase (SOD2), catalase and glutathione peroxidase (GPx) enzymatic activity levels measured 24 h later in heart, liver and lung tissues (n = 6 per experimental point). Levels of significance (P values) were determined using a Student’s two-tailed t test. Error bars represent the Standard Error of the Mean (± S.E.M.).

Fig. 3

The effects of a single 400 mg/kg dose of amifostine on SOD2, catalase and GPx enzymatic activity levels measured 24 h later in pancreas, small intestine and spleen tissues (n = 6 per experimental point). Levels of significance were determined using a two-tailed Student’s t test. Error bars = S.E.M. SOD2 activity was significantly elevated in all three tissues, while GPx activity was significantly elevated in pancreas and spleen, and catalase activity elevated only in spleen tissue.

Fig. 4

The effects of 50 mg/kg doses of amifostine administered each day for three consecutive days on enzymatic activities as a function of time in heart, liver, and lung (n = 3 per experimental time point). All analyses were performed using Stata statistical software. Significance of relevant regression parameters were tested using a two-sided significance level of 0.05. Both catalase and GPx activities were significantly elevated in heart and liver. Using this analysis, no changes in SOD2 activity reached statistical significance.

Fig. 5

The effects of 50 mg/kg doses of amifostine administered each day for three consecutive days on the subsequent changes in enzymatic activities as a function of time in pancreas, small intestine and spleen (n = 3 per experimental time point). All analyses were performed using Stata statistical software. Significance of relevant regression parameters were tested using a two-sided significance level of 0.05. Elevation of SOD2 activity reached statistical significance only in pancreas. Elevation of catalase activity reached significance in all three tissues, while elevations in GPx activity reached significance only in pancreas and spleen tissues.

Fig. 6

The effects of 50 mg/kg doses of amifostine administered each day for three consecutive days on the subsequent changes in enzymatic activities as a function of time in 8 mm diameter SA-NH tumors growing in C3H mice (n = 3 for each experimental time point). All analyses were performed using Stata statistical software. Significance of relevant regression parameters were tested using a two-sided significance level of 0.05. Elevation of SOD2, catalase and GPx enzymatic activities all reached statistically significant levels in SA-NH tumors.

Fig. 7

The effects of a single 400 mg/kg dose of amifostine to tumor-bearing C3H mice on SOD2 enzymatic activity and radiation response in SA-NH tumor cells measured 24 h later. Because amifostine is a potent radioprotector, baseline SOD2 activity and radiation response of SA-NH was measured immediately after irradiation of animals that were not exposed to the drug (n = 26 animals per experimental point) (7a). SA-NH tumor cell suspensions from 8 mm tumors grown in control and amifostine treated C3H mice were split into three aliquots, respectively, with one used for flow cytometry analysis to determine % normal cell contamination, one for the determination of colony forming efficiency in vitro, and one for an exposure to a dose of 2 Gy to determine a surviving fraction (7b). Significance was determined using a Student’s two-tailed t test. Error bars = S.E.M.